Peptide nucleic acids

Abstract

Novel peptide nucleic acids and novel linked peptide nucleic acids, form triple stranded structures with nucleic acids. The peptide nucleic acids include ligands such as naturally occurring nucleobases attached to a peptide backbone through a suitable linker. Other nucleobases including C-pyrimidines and iso-pyrimidines can be used as the ligands in Hoogsteen strands to increase binding affinity. Two peptide nucleic acid strands are joined together with a linker to form a bis-peptide nucleic acid. The individual strands of the peptide nucleic acids in the bis compounds can be orientated either parallel or antiparallel to each other.

We claim:  
     
       1. A compound comprising a first peptide nucleic acid segment and a second peptide nucleic acid segment, wherein: 
       said segments are joined via at least one linking segment;  
       said linking segment is not a peptide nucleic acid or an oligonucleotide; and  
       said peptide nucleic acid segments are of the formula:                    
       wherein: 
       n is at least 2,  
       each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands;  
       each of C 1 -C n  is (CR 6 R 7 ) y  where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 ) alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are each independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;  
       each of D 1 -D n  is (CR 6 R 7 ) where R 6  and R 7  are as defined above;  
       each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 1 but not more than 10;  
       each of G 1 -G n−1  is —NR 3 CO—, in either orientation, where R 3  is as defined above;  
       each of A 1 -A n  and B 1 -B n  are selected such that:  
       (a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N or R 3 N + ; or  
       (b) A is a group of formula (IId) and B is CH;                    
       where: 
       X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ;  
       Y is a single bond, O, S or NR 4 ;  
       each of p and q is zero or an integer from 1 to 5;  
       each of r and s is zero or an integer from 1 to 5;  
       each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and  
       each R 3  and R 4  are as defined above;  
       Q is —CO 2 H, —CONR′R″, —SO 3 H or —SO 2 NR′R″ or an activated derivative of —CO 2 H or —SO 3 H; and  
       I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.  
     
     
       2. A compound of  claim 1  wherein said linking segment includes at least one unit of an aminoalkylcarboxylic acid of the formula 
       
         
           —NH—(CH 2 ) e —C(═O)— 
         
       
       where e is 1 to 15. 
     
     
       3. A compound of  claim 2  wherein e is 4 to 8. 
     
     
       4. A compound of  claim 2  wherein said linking segment further includes at least one amino acid. 
     
     
       5. A compound of  claim 3  wherein e is 5 or 6. 
     
     
       6. A compound of  claim 1  wherein said linking segment comprises a compound of the formula 
       
         
           —(AA) h —[NH—(CH 2 ) e —C(═O)—(AA) f ] g — 
         
       
       where: 
       AA is an α-amino acid;  
       e is 4 to 8;  
       f and h are 0 or 1; and  
       g is 1 to 4.  
     
     
       7. A compound of  claim 1  wherein said linking segment includes at least one unit of a glycol amino acid. 
     
     
       8. A compound of  claim 7  wherein said glycol amino acid comprises glycol sub-units that are linked together in a linear array and that have an amino group on one terminus and a carboxyl group on the other terminus. 
     
     
       9. A compound of  claim 1  wherein said linking segment comprises a compound of the formula 
       
         
           —[NH—(CH 2 —CH 2 —O—) j —CH 2 —C(═O)—] i    
         
       
       wherein: 
       j is 1 to 6; and  
       i is 1 to 6.  
     
     
       10. A compound of  claim 9  wherein j is 2 and i is 3. 
     
     
       11. A compound of  claim 1  wherein said peptide nucleic acid segments are joined together via two of said linking segments to form a cyclic structure. 
     
     
       12. A compound of  claim 1  wherein said linking segment connects a terminal amine function on one of said first and second peptide nucleic acid segments to a carboxyl function on the other of said first and second peptide nucleic acid segments. 
     
     
       13. A compound of  claim 12  wherein said first peptide nucleic acid segment has a nucleobase sequence determined in a direction from its amine terminus to its carboxyl terminus, said second peptide nucleic acid segment has a nucleobase sequence determined in a direction from its carboxyl terminus to its amine terminus, and said sequences are the same. 
     
     
       14. A compound of  claim 1  wherein at least a portion of nucleobases of said first and second peptide nucleic acid segments are pyrimidine nucleobases. 
     
     
       15. A compound of  claim 14  wherein at least one of said pyrimidine nucleobases of one of said first or said second peptide nucleic acid segments comprises a C-pyrimidine heterocyclic base or an iso-pyrimidine heterocyclic base. 
     
     
       16. A compound of  claim 14  wherein said portion of said nucleobases that are pyrimidine nucleobases are located in contiguous homopyrimidine sequences. 
     
     
       17. A compound of  claim 1  wherein said linking segment comprises a carboxylic acid functional group and a primary amino functional group. 
     
     
       18. A compound of  claim 1  wherein said linking segment is of the formula: 
       
         
           —[HN—Z—C (═O)] n — 
         
       
       wherein: 
       n is 1 to 3; and  
       Z is C 1  to C 20  alkyl, C 2  to C 20  alkenyl, C 2  to C 20  alkynyl, C 1  to C 20  alkanoyl having at least one O or S atom, C 7  to C 34  aralkyl, C 6 -C 14  aryl or an amino acid.  
     
     
       19. A multiple stranded structure comprising: 
       a nucleic acid strand, at least a portion of which forms a target nucleotide sequence; and  
       a further strand, said further strand including first and second peptide nucleic acid segments that are joined together via a linker;  
       wherein: 
       said first peptide nucleic acid segment has a nucleobase sequence that is complementary to the target nucleotide sequence in the 5′ to 3′ direction of said target nucleotide sequence;  
       said second peptide nucleic acid segment has a nucleobase sequence that is complementary to the target nucleotide sequence in the 3′ to 5′ direction of said target nucleotide sequence; and  
       said peptide nucleic acid segments are of the formula:                    
       wherein: 
       n is at least 2,  
       each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, provided that at least one of said L 1 -L n  is a C-pyrimidine heterocyclic base or an iso-pyrimidine heterocyclic base;  
       each of C 1 -C n  is (CR 6 R 7 ) y  where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are each independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;  
       each of D 1 -D n  is (CR 6 R 7 ) z  where R 6  and R 7  are as defined above;  
       each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 1 but not more than 10;  
       each of G 1 -G n−1  is —NR 3 CO—, in either orientation, where R 3  is as defined above;  
       each of A 1 -A n  and B 1 -B n  are selected such that:  
       (a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N or R 3 N + ; or  
       (b) A is a group of formula (IId) and B is CH;                    
       where: 
       X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ;  
       Y is a single bond, O, S or NR 4 ;  
       each of p and q is zero or an integer from 1 to 5;  
       each of r and s is zero or an integer from 1 to 5;  
       each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and  
       each R 3  and R 4  are as defined above;  
       Q is —CO 2 H, —CONR′R″, —SO 3 H or —SO 2 NR′R″ or an activated derivative of —CO 2 H or —SO 3 H; and  
       I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.  
     
     
       20. The structure of  claim 19  wherein said nucleic acid strand is a single stranded DNA or RNA. 
     
     
       21. The structure of  claim 19  wherein said nucleic acid strand is a double stranded DNA. 
     
     
       22. The structure of  claim 19  wherein one of said first or second peptide nucleic acid segments exhibits Watson/Crick binding to said target nucleotide sequence and the other of said first and second peptide nucleic acid segments exhibits Hoogsteen binding to said target nucleotide sequence. 
     
     
       23. The structure of  claim 22  wherein said one of said first or second peptide nucleic acid segments that exhibits Hoogsteen binding to said target nucleotide sequence includes C-pyrimidine heterocyclic nucleobases or iso-pyrimidine heterocyclic nucleobases in at least one of the positions that are complementary to nucleobases in said target nucleotide sequence. 
     
     
       24. The structure of  claim 23  wherein said C-pyrimidine heterocyclic nucleobase or iso-pyrimidine heterocyclic nucleobase is pseudo-isocytosine, iso-cytosine, pseudo-uracil or 5-bromouracil. 
     
     
       25. A compound comprising a peptide nucleic acid strand of the formula:                    
       wherein: 
       n is at least 2,  
       each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, provided that at least one of said L 1 -L n  is pseudo-uracil;  
       each of C 1 -C n  is (CR 6 R 7 ) y  where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are eac independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;  
       each of D 1 -D n  is (CR 6 R 7 ) 2  where R 6  and R 7  are as defined above;  
       each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 1 but not more than 10;  
       each of G 1 -G n−1  is —NR 3 CO—, in either orientation, where R 3  is as defined above;  
       each of A 1 -A n  and B 1 -B n  are selected such that:  
       (a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N or R 3 N + ; or  
       (b) A is a group of formula (IId) and B is CH;                    
       where: 
       X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ;  
       Y is a single bond, O, S or NR 4 ;  
       each of p and q is zero or an integer from 1 to 5;  
       each of r and s is zero or an integer from 1 to 5;  
       each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and  
       each R 3  and R 4  are as defined above;  
       Q is —CO 2 H, —CONR′R″, —SO 3 H or —SO 2 NR′R″ or an activated derivative of —CO 2 H or —SO 3 H; and  
       I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.  
     
     
       26. A compound comprising a peptide nucleic acid strand of the formula:                    
       wherein: 
       n is at least 2,  
       each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, provided that at least one of said L 1 -L n  is 5-bromouracil;  
       each of C 1 -C n  is (CR 6 R 7 ) y  where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are each independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;  
       each of D 1 -D n  is (CR 6 R 7 ) z  where R 6  and R 7  are as defined above;  
       each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 1 but not more than 10;  
       each of G 1 -G n−1  is —NR 3 CO—, in either orientation, where R 3  is as defined above;  
       each of A 1 -A n  and B 1 -B n  are selected such that:  
       (a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N or R 3 N + ; or  
       (b) A is a group of formula (IId) and B is CH;                    
       where: 
       X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ;  
       Y is a single bond, O, S or NR 4 ;  
       each of p and q is zero or an integer from 1 to 5;  
       each of r and s is zero or an integer from 1 to 5;  
       each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and  
       each R 3  and R 4  are as defined above;  
       Q is —CO 2 H, —CONR′R″, —SO 3 H or —SO 2 NR′R″ or an activated derivative of —CO 2 H or —SO 3 H; and  
       I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.